The potential of statin and stromal cell-derived factor-1 to promote angiogenesis

Angiogenesis requires the mobilization of progenitor cells from the bone marrow (BM) and homing of progenitor cells to ischemic tissue. The cholesterol lowering drug Statins can stimulate angiogenesis via mobilization of BM derived endothelial progenitor cells (EPCs), promoting EPC migration, and inhibiting EPC apoptosis. The chemokine stromal cell-derived factor-1 (SDF-1) augments EPC chemotaxis, facilitates EPC incorporation into the neovasculature. The combined use of a statin to mobilize EPCs and local overexpression of SDF-1 to augment EPC homing to ischemic muscle resulted in superior angiogenesis versus use of either agent alone. Their effects are through augmenting EPC mobilization, incorporation, proliferation, migration and tube formation while inhibiting EPC apoptosis. Statin and SDF-1 therefore display synergism in promoting neovascularization by improving reperfusion of ischemic muscle, increasing progenitor cell presentation and capillary density in ischemic muscle, and diminishing apoptosis. These results suggest that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.Key words: angiogenesis, endothelial progenitor cells, statin, SDF-1, migrationAngiogenesis is the process by which new vessels form in ischemic tissue. The cytokine Stromal Cell Derived Factor-1 (SDF-1) is released into the circulation in response to ischemia and is an initiating signal in the angiogenesis process. SDF-1 mobilizes bone marrow cells (BMC) by binding to the cell surface receptor CXCR4. BMCs then enter the circulation and migrate to the ischemic site following the SDF-1 gradient. On arrival, BMCs promote angiogenesis by providing cellular elements such as endothelial cells (EC) and perivascular cells and also by secreting signaling proteins that mature the angiogenesis process. BMC surface CXCR4 expression and the SDF-1/CXCR4 interaction are essential for BMC to home to the injured site.Cell-based strategies to improve neovascularization of ischemic tissue have been achieved by injecting mononuclear cells derived from either BM¹ or peripheral blood, directly into ischemic muscle,² or by mobilizing BM-MNC with cytokines³ or other drugs such as statins.^4–6Statins are 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors and are primarily used to lower circulating cholesterol levels. In addition to reducing cholesterol synthesis, inhibition of the mevalonate pathway prevents synthesis of isoprenoid intermediates including geranylgeranylpyrophosphate. Geranylgeranylation is important in the posttranslational modification of intracellular signaling proteins, including Rho GTPases. This mechanism underlies many of the pleiotropic effects including the ability of statins to stabilize endothelial nitric oxide synthase mRNA and increase nitric oxide biosynthesis. In fact, statins have been shown to protect against ischemic injury of the heart and stimulate angiogenesis in ischemic limbs of normocholesterolemic animals.^7,8 The mechanism of action of statins has been demonstrated via mobilization of BM endothelial progenitor cells (EPCs) and facilitation of EPC incorporation into the neovasculature through a phosphoinositide-3 (PI-3) kinase-dependent pathway.^4–6 Statins have also been reported to enhance EPC migration, augment EPC chemotaxis and inhibit EPC apoptosis both in vitro and in vivo.^4,9,10SDF-1, an 89-amino acid polypeptide, is a member of the chemokine CXC subfamily originally isolated from murine bone marrow stromal cells.¹¹ SDF-1 was initially identified as a potent chemoattractant for lymphocytes and monocytes, and as an enhancer of B cell proliferation. SDF-1 is considered to be a key regulator of hematopoietic stem cell trafficking between BM and the peripheral circulation. SDF-1 is highly expressed in ischemic tissues.^12,13 Elevation of SDF-1 levels in peripheral blood results in BMC mobilization to the peripheral circulation with a concurrent decrease within the BM.¹⁴ SDF-1 not only mobilizes progenitor cells in BM but also directs them to the ischemic site by promoting cell migration and proliferation.^3,15 SDF-1 may generate a gradient similar to developmental morphogens during ischemia that provides the cues and directions for progenitor cell mobilization into peripheral blood and homing to ischemic tissues.^16,17 Furthermore, SDF-1 also reduces EPC apoptosis and enhances survival of the progenitor cells.^3,18 SDF-1, either delivered locally in its protein form,^3,19,20 or generated in situ via plasmid and viral vector-mediated gene expression,^10,21,22 enhances neovascularization by augmenting EPC recruitment into ischemic tissues.SDF-1 binding to its receptor CXCR4 on the cell surface provides essential signals for mobilization and homing of EPCs to the injured site.^23–25 SDF-1 binding with CXCR4 triggers internalization of CXCR4. This SDF-1/CXCR4 interaction results in elevation of cytoplasmic Ca²⁺ levels²⁶ and phosphorylation of PI-3 kinase and other protein kinases, e.g. Akt,²¹ MEK/ERK^27,28 and Janus kinase (JAK)-2.²⁹ Activation of Akt protein kinase further upregulates the activity of eNOS by increasing both eNOS expression and phosphorylation, which in turn catalyzes the production of nitric oxide (NO), an important signal molecule for vascular protection and remodeling.^21,26 Disruption of SDF-1/CXCR4 interaction impaired incorporation of EPC into sites of ischemia, and disturbed ischemic limb neo-vascularization.³⁰To explore if the combined use of a statin to mobilize BM EPCs and local overexpression of SDF-1 to augment EPC homing to ischemic muscle will result in superior angiogenesis versus use of either agent alone, we used the murine hindlimb ischemia model to determine the effects of Fluvastatin and SDF-1 on angiogenesis.¹⁰ Fluvastatin (5 mg/kg) was injected intra-peritoneally into the mice daily for 7 days to mobilize progenitor cells prior to ischemia-inducing surgery. NIH 3T3 cells transduced with the retroviral vector carrying SDF-1 gene were injected I.M. into the ischemic limb after surgery to locally deliver SDF-1 to ischemic muscle.²² The number of circulating EPCs increased 9–18 fold seven days post statin/SDF-1 treatment.Our data of single treatment with Fluvastatin are consistent with the previous reports that statins not only augment mobilization of progenitor cells by increasing circulating EPC originated from BM,^4,31 but also modulate their differentiation. We further give a new insight view of the mechanism for statin induced EPC mobilization. We found that statin induced activation of matrix metalloproteinases (MMP)-2 and -9 in EPC. The increased MMP activity could result in degradation of extracellular matrix.¹⁷ Progenitor cells will be such mobilized into circulation when the cellular attachment is reduced within the bone marrow niches. We show that statin alone can enhance the phosphorylation of Akt, promote EPC proliferation, migration and inhibit cell apoptosis in vitro. The proangiogenic effects of statin are also illustrated in vivo using a murine hind-limb ischemia model. In this model, Fluvastatin treatment results in more EPC in circulation, more BM derived progenitor cells in ischemic muscle, more cell proliferation, enhanced capillary formation, and diminished cell apoptosis; these effects end up in improved reperfusion versus control. The beneficial effects of statin on angiogenesis are independent of cholesterol since the total serum cholesterol level is not changed by Fluvastatin treatment under these experimental conditions.To be noted, the effect of statins on EPCs was found to be concentration dependent. EPC proliferation, migration and the inhibition of apoptosis are enhanced at low statin concentrations (10 nM and 100 nM) but are significantly inhibited at a higher statin concentration (1,000 nM). The toxic effect of statin at high concentration cannot be compensated by addition of SDF-1, indicating that Statin causes apoptosis in a pathway different from the pathway that SDF-1 uses to prevent EPC apoptosis. Increased apoptosis at the higher statin concentration could explain the reversed effect of stain in angiogenesis. These findings are consistent with the reports in which statins were found to have proangiogenic effects at low therapeutic concentrations but angiostatic effects at high concentrations, the latter effect being reversible by geranylgeranyl pyrophosphate.^32,33Combined statin and SDF-1 treatment significantly enhanced angiogenesis versus treatment with either reagent alone. More cell proliferation and less apoptosis were observed both in vitro and in vivo, along with increased cell migration and tube formation in vitro, and enhanced progenitor cell incorporation and higher capillary density in ischemic tissue in vivo. It is interesting to note that neither statin nor SDF-1 alone promotes EPC tube formation, but combined treatment results in significant EPC tube formation. These results suggest that SDF-1 and statin have different mechanisms of action with regards to the promotion of neovascularization. It is possible that each drug affects a specific subset of progenitor cells.The facilitative effect of both statin and SDF-1 on EPC proliferation and migration is involved with Akt phosphorylation and endothelial nitric oxide synthase (eNOS) activation. The mechanism by which statins promote angiogenesis is through, at least partly, improved nitric oxide bioavailability. Statins have been reported to induce eNOS mRNA stability³⁴ and eNOS activity through a PI3k/Akt dependent pathway.^31,35–37 However, neither eNOS mRNA/protein expression nor EPCs are reported to be essential for the therapeutic effect of Fluvastatin on hypoxia-induced pulmonary hypertension; Fluvastatin improved eNOS phosphorylation by a mechanism independent of Akt activation.³⁸ Our data favor a mechanism involving Akt phosphorylation since phosphorylated Akt is increased when EPCs are cultured in the presence of statin, and statin-enhanced EPC proliferation and migration were inhibited by the PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.The angiogenic effects of SDF-1 also involve increased production of NO²⁶ as NO is essential for EC migration and angiogenesis. SDF-1α gene transfer has been shown to enhance eNOS activity.²¹ Our in vitro data confirmed the involvement of Akt and eNOS in SDF-1 mediated cell migration.¹⁰ Phosphorylated Akt is increased when EPCs are cultured in the presence of SDF-1. The facilitative effect of SDF-1 on EPC migration is blocked by both the Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the eNOS inhibitor L-NMMA. In contrast, L-NMMA does not reverse the inhibitory effect of SDF-1 on apoptosis, indicating that the inhibitory effect of SDF-1 on apoptosis is not mediated through NO.²²We also show that the expression of MMP-2 and MMP-9 was increased when EPCs were cultured in the presence of statin or SDF-1. MMPs are a family of proteolytic enzymes that degrade components of the extracellular matrix (ECM). Degradation of ECM is an essential step for cell mobilization and migration. Our data indicate that the novel effect of statin and SDF-1 on migration is through enhancement of MMP-2 and MMP-9 activity, resulting in ECM degradation, thus promoting progenitor cell mobilization and migration. Both Akt phosphorylation and expression of MMP-2 and MMP-9 in EPCs are further enhanced by combined treatment with statin and SDF-1. This result indicates that treatment of EPCs with either statin or SDF-1 as monotherapy results in a sub-maximal angiogenic response. The effects of statin partially overlap with that of SDF-1; and the combined use of two factors appears to have an optimal effect on progenitor cells (Fig. 1).Open in a separate windowFigure 1Effect of statins and SDF-1 on promoting angiogenesis. Statin enhances the phosphorylation of Akt with a yet undefined mechanism. SDF-1 binding with the G-protein coupled membrane receptor CXCR4 results in phosphorylation of protein kinases like PI3 kinase and Akt. Activation of Akt then upregulates the activities of MMPs and eNOS. NOS catalyze the synthesis of NO which is essential for the EPC migration. MMPs degrade extracellular matrix to initiate cell migration. Activation of Akt also prevents cell apoptosis. These reactions promote cell migration and proliferation and enhance EPC survival. EPCs from bone marrow are thus mobilized into circulation. The circulating EPC are homed into ischemia area in lure of SDF-1. EPCs contribute to neovascularisation, either directly by incorporation into endothelium and differentiation into endothelial cells or indirectly by differentiating into perivascular cells that provide physical support and secrete signaling proteins and structural enzymes enabling the angiogenesis process. The effects of statin partially overlap with that of SDF-1; and the combined use of two factors appears to have an additive/synergistic effect on progenitor cells.In summary, the combination of progenitor cell mobilization with statin and targeted recruitment into the ischemic bed by SDF-1 leads to improved blood flow in the ischemic limb versus treatment with either agent alone. Statin and SDF-1 therefore display synergism in promoting neovascularization. This result suggests that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia. However, the use of statins as a clinical modifier of angiogenesis is still unproven. A great number of patients have been treated with these drugs and if they were potently proangiogenic, one might expect to see an increased risk of tumors. However, there is no evidence that these drugs encourage tumor development. Likewise, there is no definitive evidence for an antiangiogenic, tumor-modulating action of statins. We await further studies with interest.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Myosin light chain kinases: Division of work in cell migration

Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.Key words: myosin, MLCK, ROK, MRCK, phosphorylation, cell migrationCell movement is a fundamental activity underlying many important biological events ranging from embryological development to immunological responses in the adult. A typical cell movement cycle entails polarization, membrane protrusion, formation of new adhesions, cell body translocation and finally rear retraction.¹ A precise temporal and spatial coordination of these separate steps that take place in different parts of the cell is important for rapid and efficient movement.²One major event during eukaryotic cell migration is the myosin II (MYO2)-mediated contraction that underlies cell protrusion, traction and retraction.^1,3 An emerging theme from collective findings is that there are distinct myosin contractile modules responsible for the different functions which are separately regulated by local myosin regulatory light chain (MLC) kinases. These kinases contribute to contractile forces that connect adhesion, protrusion and actin organization.² Unraveling the regulation of these contractile modules is therefore pivotal to a better understanding of the coordination mechanism.At the lamellipodium, the conventional calcium/calmodulin-dependent myosin light chain kinase (MLCK) has been shown to play an essential role in a Rac-dependent lamellipodial extension.⁴ Inhibition of calmodulin or MLCK activity by specific photoactivatable peptides in motile eosinophils effectively blocks lamellipodia extension and net movement.⁵ Furthermore, there is a strong correlation between activated MLCK and phosphorylated MLC within the lamellipodia of Ptk-2 cells as revealed by fluorescence resonance energy transfer (FRET) analysis.⁶ More recent studies showed MLCK to regulate the formation of focal complexes during lamellipodia extension.^7,8 Functionally, MLCK is thought to play a critical role in the environment-sensing mechanism that serves to guide membrane protrusion. It mediates contraction that exerts tension on integrin-extracellular matrix (ECM) interaction, which, depending on the rigidity of the substratum, will lead to either stabilization of adhesion resulting in protrusion or destabilization of attachment seen as membrane ruffling on non-permissive surfaces.^8,9As a Rho effector, Rho-associated kinase (ROK/ROCK/Rho-kinase) has been shown to regulate stress fibers and focal adhesion formation by activating myosin, an effect that can be blocked by the specific ROK inhibitor Y-27632.^10,11 Myosin activation by ROK is the effect of two phosphorylation events: the direct phosphorylation on MLC and the inhibition of myosin phosphatase through phosphorylation of its associated myosin-binding subunit (MBS).¹¹ Consistent with this notion of a localization-function relationship, ROK and MBS, which can interact simultaneously with activated RhoA,¹¹ have been shown to colocalize on stress fibers.^12,13 In migrating cells, Rho and ROK activities have been mostly associated with the regulation of tail retraction, as inhibition of their activities often results in trailing tails due to the loss of contractility specifically confined to the cell rear.^14,15 Tail retraction requires high contractile forces to overcome the strong integrin-mediated adhesion established at the rear end, an event which coincides with the strategic accumulation of highly stable and contractile stress fibers that assemble at the posterior region of migrating cells.MRCK was previously shown to phosphorylate MLC and promote Cdc42-mediated cell protrusion.¹⁶ More recently, it was found to colocalize extensively with and regulate the dynamics of a specific actomyosin network located in the lamella and cell center, in a Cdc42-dependent manner but independent of MLCK and ROK.¹⁷ The lamellar actomyosin network physically overlaps with, but is biochemically distinct from the lamellipodial actin meshwork.^9,18 The former network consists of an array of filaments assembled in an arrangement parallel to the leading edge, undergoing continuous retrograde flow across the lamella, with their disassembly occurring at the border of the cell body zone sitting in a deeper region.^17–19 Retrograde flow of the lamellar network plays a significant role in cell migration as it is responsible for generating contractile forces that support sustained membrane protrusion and cell body advancement.^17–19It is therefore conceivable that these three known MLC kinases are regulated by different signaling mechanisms at different locations and on different actomyosin contractile modules. The coordination of the various modules will ensure persistent directional migration (Figure 1). Phosphorylation of MLC by PAK and ZIP kinase has also been reported, but their exact roles in this event have yet to be determined.^20,21 It is also noteworthy that individual kinases can work independently of each other, as amply shown by evidence from inhibitor treatments. This is particularly true for MRCK in the lamella, whose activity on lamellar actomyosin flow is not affected by ML7 and Y-27632, the inhibitors of MLCK and ROK respectively.¹⁷ These findings further indicate that although both ROK and MRCK have been shown to upregulate phosphorylated MLC levels by inhibiting the myosins phosphatases,^11,22 they are likely to act as genuine MLC kinases themselves, without the need of MLCK as previously suggested.¹¹Open in a separate windowFigure 1Upper panel depicts a model for the specific activation of the different MLC kinases at various locations in the cell. In response to upstream signals, MLC kinases MLCK, MRCK and ROK are activated and localized to different regions. In the case of MRCK and ROK, the interaction of the GTP-bound Rho GTPase binding domain will determine the specific action of the downstream kinase, resulting in actomyosin contractility at different locations. The coordination of these signalling events is crucial for directional cell migration. Lower panel shows a typical front-rear location for Myosin 2A and 2B in a migrating U2OS cell.In conjunction with their differences in localization, the three MLC kinases show apparent individual preferences and specificity towards the MYO2 isoforms that they associate with. The two major MYO2 isoforms MYO2A and 2B are known to have distinct intracellular distributions that are linked to their individual functions (Figure 1).^23,24 In motile cells, MYO2A localization that is skewed towards the protruding cell front is consistent with it being the major myosin 2 component of the lamellar filaments regulated by MRCK as well as its regulation by MLCK in lamellipodial contraction.^8,17,19 In contrast, the enrichment of MYO2B at retracting cell rear conforms well with the requirement of thick and stable stress fibers capable of causing tail contraction and prevention of protrusion under the control of Rho/ROK signaling.^23,25 The selection for MYO2B filaments in the cell rear stems from their more contractile and stable nature compared with MYO2A, a consequence of their higher time-averaged association with actin.^26,27 Conversely, the lower tension property of MYO2A filaments suggests that they are more dynamic in nature,^26,27 a characteristic which fits well with the dynamic actomyosin activities at the leading edge and lamella that regulate protrusion.It deserves special mention that the three MLC kinases display subtle differences in their specificity towards MLC. While MLCK and MRCK phosphorylate only a single Ser19 site (monophosphorylation),^18,28 ROK is able to act on both Thr18 and Ser19 residues causing diphosphorylation of MLC,²⁹ MLCK only causes diphosphorylation when present at higher concentrations.³⁰ By further increasing its actin-activated ATPase activity, diphosphorylation of MLC has been shown to induce a higher myosin activation and filament stability.^30–32 The use of specific antibodies that can differentiate between the two populations of phosphorylated MLC has been instrumental in revealing their localization and correlation with the activity of the MLC kinases. The emerging picture from these experiments is that mono and diphosphorylated MLC exhibit distinct distributions in migrating cells, with the monophosphorylated MLC localized more towards the protrusive region, while the diphosphorylated form is more enriched at the posterior end.^21,33 Taking into account their biochemical properties, the polarized distributions of these differentially phosphorylated MLC coincide functionally with the segregation of the MYO2 isoforms and their corresponding regulators. These findings provide further support for the existence of segregated contractile modules in migrating cell and their distinctive regulation.The mechanisms that determine the specific segregation of the contractile modules and their regulation are unclear. However, some clues have emerged from recent studies. It has been shown that the C-terminal coiled-coil region of MYO2B is important for determining its localization in cell rear²⁵ and which requires Rho/ROK activity as their inhibition resulted in the loss of this specific localization.²³ Correspondingly, the inhibition of MRCK activity resulted in the loss of lamella-localized MYO2A.¹⁷ These findings suggest that activation of MYO2 filaments by their upstream regulators is important for their functional segregation and maintenance. It is noteworthy that both ROK and MRCK have distinct regulatory domains including the pleckstrin homology domains which have been shown to be essential for their localization, a process which may involve myosin interaction and lipid-dependent targeting as has been respectively shown for ROK and MRCK.^11,13,16 Further, the specificity of MRCK for lamellar actomyosin is believed to be largely determined by the two proteins it forms a complex with: the adaptor LRAP35a, and the MYO2-related MYO18A. Activation of MYO18A by MRCK, a process bridged by LRAP35a, is a crucial step which facilitates MRCK regulation on lamellar MYO2A.¹⁷The mechanisms responsible for segregating the contractile modules and their regulators may also comprise a pathway that parallels the microtubule-modulatory Par6/aPKC/GSK3β signalling pathway which regulates cellular polarization. This notion is supported by both Cdc42 and Rho being common upstream regulators of these two pathways.³⁴ GTPase activation may determine the localized activities of the separate contractile modules and create an actomyosin-based asymmetry across the cell body, which together with the microtubule-based activities, result in the formation of a front-back axis important for directional movement. The involvement of MRCK in MTOC reorientation and nuclear translocation events,³⁵ and our unpublished observation that LRAP35a has a GSK3β-dependent microtubule stabilizing function are supportive of a possible cross-talk between these two pathways.In conclusion, the complex regulation of contractility in cell migration emphasizes the importance of the localization, specificity and enzymatic properties of the different MLC kinases and myosin isoforms involved. The initial excitement and confusion caused by the emergence of the different MLC kinases are fading, being now overtaken by the curiosity about how they cooperate and are coordinated while promoting cell motility.     

The Neurofibromatosis 2 Protein,Merlin, Regulates Glial Cell Growth in an ErbB2- and Src-Dependent Manner

Individuals with the inherited cancer predisposition syndrome neurofibromatosis 2 (NF2) develop several central nervous system (CNS) malignancies, including glial cell neoplasms (ependymomas). Recent studies have suggested that the NF2 protein, merlin (or schwannomin), may regulate receptor tyrosine kinase signaling, intracellular mitogenic growth control pathways, or adherens junction organization in non-nervous-system cell types. For this report, we used glial fibrillary acidic protein conditional knockout mice and derivative glia to determine how merlin regulates CNS glial cell proliferation. We show that the loss of merlin in glial cells results in increased proliferation in vitro and in vivo. Merlin regulation of glial cell growth reflects deregulated Src activity, such that pharmacologic or genetic inhibition of Src activation reduces Nf2^−/− glial cell growth to wild-type levels. We further show that Src regulates Nf2^−/− glial cell growth by sequentially regulating FAK and paxillin phosphorylation/activity. Next, we demonstrate that Src activation results from merlin regulation of ErbB2 activation and that genetic or pharmacologic ErbB2 inhibition reduces Nf2^−/− glial cell Src/Src effector activation and proliferation to wild-type levels. Lastly, we show that merlin competes with Src for direct binding to ErbB2 and present a novel molecular mechanism for merlin regulation of ErbB2-dependent Src signaling and growth control.Neurofibromatosis type 2 (NF2) is an autosomal dominant inherited cancer syndrome in which affected individuals develop nervous system tumors, including peripheral nerve tumors (schwannomas), leptomeningeal tumors (meningiomas), and glial fibrillary acidic protein (GFAP)-immunoreactive glial cell tumors (spinal ependymomas). NF2 results from a germ line mutation in the NF2 tumor suppressor gene, located on chromosome 22q (46, 60). Tumors in this disorder arise following somatic inactivation of the one remaining wild-type (WT) NF2 allele in specific cell types. In this regard, NF2-associated schwannomas, meningiomas, and ependymomas all exhibit biallelic NF2 gene inactivation (33, 47, 61). In addition, NF2 gene inactivation is also observed in 50 to 78% of sporadic schwannomas, 32 to 84% of sporadic meningiomas, and 37% of sporadic ependymomas (21, 29), suggesting that this gene is also a key growth regulator in nonhereditary nervous system cancers.The NF2 gene was identified in 1993 and found to code for a 595-amino-acid protein, termed merlin or schwannomin (46, 60). Analysis of the predicted protein sequence revealed striking sequence similarity between merlin and a family of protein 4.1 family members that link the actin cytoskeleton to cell surface glycoproteins (55). In particular, merlin most closely resembles the ezrin/radixin/moesin (ERM) subfamily and has been shown to bind actin as well as to associate with several cell surface glycoproteins, including CD44 and β1-integrin (5, 32, 48). However, unlike the ERM proteins, merlin is unique in its capacity to function as a nervous system tumor suppressor gene.In order to identify the key signaling pathways regulated by the merlin tumor suppressor protein, previous studies have focused on merlin growth regulation in fibroblasts, primary Schwann cell and human schwannoma cell cultures, meningioma and schwannoma tumor cell lines, and other non-central nervous system (non-CNS) cell types. These investigations have resulted in the identification of a large number of nonintersecting growth control pathways regulated by merlin in different cell types. In this regard, merlin has been implicated in epidermal growth factor receptor (EGFR) (9), β1-integrin (15), and CD44 (1, 35, 48) function as well as in Ras (25, 59), Rac1 (34, 52), phosphatidylinositol 3-kinase (44), mitogen-activated protein kinase (MAPK) (7, 30), and STAT (51) intracellular signaling. While each of these pathways is involved in growth control in the brain, it is not known which of these intracellular signaling pathways are deregulated in NF2-deficient CNS cell types.To gain insights into the role of the NF2 gene in glial cell growth control relevant to the development of targeted therapies for NF2-associated glial cell malignancies, we studied the consequence of merlin loss on the growth of primary brain glial cells (astrocytes) in vitro and in vivo, using Nf2 conditional knockout genetically engineered mice (GEM). We demonstrate for the first time that merlin regulates brain glial cell growth by controlling the phosphorylation/activity of Src and its downstream effectors, FAK and paxillin. Furthermore, we show that merlin regulation of Src phosphorylation/activation is modulated by ErbB2 phosphorylation/activation and ErbB2-Src binding. Finally, we show that merlin competitively inhibits Src binding to ErbB2 and, in this manner, prevents ErbB2-mediated Src phosphorylation and downstream mitogenic signaling. Based on these findings, we propose a novel mechanism for merlin growth regulation in CNS glia.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

A herpesvirus kinase that masquerades as Akt: You don't have to look like Akt,to act like it

The cellular protein synthesis machinery is tightly regulated and capable of rapid reaction to a variety of physiological inputs critical in stress-response, cell cycle control, cancer biology and virus infection. One important strategy for stimulating protein synthesis involves the ser/thr kinase Akt, which subsequently triggers inactivation of the cap-dependent translational repressor 4E-BP1 by an mTOR-containing protein complex (mTORC1). A recent paper demonstrated that herpes simplex virus utilizes a remarkable tactic to activate mTOR in infected cells. Instead of using the cellular Akt, the virus produces a ser/thr kinase called Us3 that doesn''t look like Akt, but masquerades as Akt. By making the Akt-like protein unrecognizable, this disguise allows it to bypass the strict limits normally imposed on the real cellular Akt. Importantly, preventing the virus Akt-imposter from triggering mTORC1 inhibited viral growth, suggesting a new way to block herpes simplex virus. This study also raises the possibility that other Akt-impersonators may lurk hidden in our own genomes, possibly contributing to diseases ranging from diabetes to cancer.Key words: Akt signaling, translational control, mTORC1 activation, virus replication, viral kinaseBy manipulating activity of the translation repressor 4E-BP1, Akt signaling through mTORC1 controls a critical step regulating the initiation of protein synthesis in eukaryotes.¹ 4E-BP1 represses translation by binding to the cellular cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E), preventing its incorporation into the eIF4F multi-subunit complex required to initiate translation.² Hyperphosphorylation of 4E-BP1 by mTORC1 inactivates the repressor, allowing eIF4E to associate with the large molecular scaffold eIF4G and assemble eIF4F (Fig. 1).¹ Subsequently, hyperphosphorylated 4E-BP1 can be degraded by the proteasome.^3–5 Regulating assembly of eIF4F is a major point through which 40S ribosome subunits are recruited and loaded onto the mRNA 5′ end. Indeed, numerous biological regulatory processes where differential control of translation plays a fundamental role often involve 4E-BP1 hyperphosphorylation by mTORC1.⁶Open in a separate windowFigure 1Repression of eIF4F assembly and cap-dependent mRNA translation by 4E-BP1 phosphorylation. The cellular cap-binding protein (4E) is depicted bound to the translational repressor eIF4E-binding protein 1 (4E-BP1) and is unable to assemble into an eIF4F complex with the other translation initiation factors, eIF4G and eIF4A. Activation of the kinase mTOR in response to a variety of cues such as HSV-1 infection, growth factor signaling, alterations to the nutrient pool or changes in cellular energy reserves results in phosphorylation of the translational repressor protein 4E-BP1 and the release of eIF4E from 4E-BP1. Binding of eIF4E to eIF4G and eIF4A results in assembly of the multisubunit initiation factor eIF4F complex, which in turn recognizes the 7-methyl guanine cap (m⁷) at the mRNA 5′ end. The 40S ribosome is recruited through its association with eIF3.Diverse inputs including nutrient, energy and growth factor availability are integrated by the tuberous sclerosis heterodimer complex (TSC1/2), which controls mTORC1 activation.¹ TSC is a GTPase activating protein (GAP) for the small G-protein rheb (ras-homolog enriched in brain). Whereas rheb•GTP activates mTORC1, rheb•GDP cannot. Thus, TSC GAP activation results in rheb•GDP accumulation and inactive mTORC1, while TSC GAP inhibition results in rheb•GTP accumulation and stimulates mTORC1 (Fig. 2). TSC activity is controlled by phosphorylation of the TSC2 subunit by different cellular kinases. One of these is Akt. In response to PI3-kinase activation, recruitment of PDK1 and Akt to the plasma membrane results in Akt phosphorylation at T308. Fully active Akt results after a second mTOR-containing complex, mTORC2, phosphorylates S473. Phosphorylation of TSC2 by Akt on S939 and T1462 inhibits TSC Gap activity and thereby activates mTORC1. Although mTORC1 can be regulated by inputs from other signaling pathways including Erk/RSK, which also can phosphorylate TSC2 to inhibit its GAP function and Rag-GTPases, which are required for mTORC1 activation in response to amino acids, Akt signaling is required for growth factors and hormone-responsive mTORC1 activation and the resulting stimulation of protein synthesis.⁷ In addition to TSC2 phosphorylation, Akt can also phosphorylate and inactivate PRAS40, an mTORC1 inhibitory subunit.⁸ Phosphorylation of the mTORC1 substrates ATG1, ribosomal protein S6 kinase and 4E-BP1 regulate autophagy, cell size, cell proliferation and protein synthesis.^9,10 In addition, inhibition of mTORC1 with rapamycin decelerates cellular senescence.¹¹ These basic processes are important in a variety of pathophysiological settings, including response to stress, cell cycle control, age-related diseases, cancer biology and virus infection.Open in a separate windowFigure 2Phosphorylation of TSC2 by Akt or Us3 activates mTORC1. Growth factor-mediated activation of mTORC1 is illustrated. IRS1 is recruited to the cytoplasmic face of activated growth factor receptors and stimulates PI3-kinase signaling. PDK1 and Akt are both localized to the plasma membrane via a lipid-binding plekstrin homology domain. Upon activation by PI3-kinase, PDK1 phosphorylates Akt on T308, thereby contributing to Akt activation. Full Akt activation also requires S473 phosphorylation by mTORC2. Akt inhibits TSC rheb-GAP activity by phosphorylating TSC2 on T1462/S939, promoting rheb•GTP-mediated mTORC1 activation and subsequent 4E-BP1 hyperphosphorylation and activation of p70 S6K (S6K). Inactivation of the translational repressor 4E-BP1 promotes binding of eIF4E to eIF4G, stimulating cap-dependent translation. An intrinsic feedback control circuit whereby inactivation of IR S1 and mTORC2 by activated p70 S6K limits Akt activation. Even though it is unrelated to Akt at the primary sequence level, the HSV1 ser/thr kinase Us3 phosphorylates TSC2 on the same residues targeted by Akt (T1462, S939). By targeting TSC2, this strategy allows Us3 to bypass the intrinsic feedback controls designed to limit Akt activation, allowing HSV1 to constitutively inactivate 4E-BP1 and maintain high levels of viral mRNA translation in infected cells.Following lytic infection of a host cell or reactivation from latency, herpesviruses stimulate the cap-dependent translation machinery of their hosts by promoting eIF4F assembly.^3,12–15 This is often a critical step in the virus replication cycle, as viruses are completely dependent on the translation machinery resident in their cellular hosts to produce viral proteins required for their productive growth. Similar to uninfected cells, herpesvirus-induced eIF4F assembly was sensitive to recently-developed mTOR active site inhibitors and impaired by expression of a dominant 4E-BP1 repressor allele with T→A substitutions at the key T37 and T46 sites.^16–18 However, in a major departure from findings in uninfected cells, 4E-BP1 was constitutively hyperphosphorylated in HSV-1-infected cells in the presence of allosteric Akt inhibitors.¹⁷ Precisely how HSV-1 was able to stimulate mTORC1 in the absence of Akt signaling was not known.Recently, we established that the HSV-1 ser/thr kinase encoded by the Us3 gene is required to activate mTORC1, inactivate the 4E-BP1 translational repressor and stimulate eIF4F assembly. Surprisingly, Us3 displays no sequence homology with the cellular kinase Akt, yet directly phosphorylates tuberous sclerosis complex 2 (TSC2) on S939 and T1462, the same sites targeted by Akt to inhibit TSC activity and activate mTORC1 in uninfected cells.¹⁷ While it is not unusual for virus infection to stimulate Akt signaling, this typically involves PI3-kinase activation by a virus-encoded gene product, such as Influenza virus A NS1, HSV1 VP11/12, KSHV-encoded GPCR, HCMV IE1/2, EBV LMP2A and Adenovirus E4 orf1 or a less well-understood protein phosphatase 2A-dependent process involving HPV E7 or Adenovirus E4 orf4 that may prevent dephosphorylation of mTORC1 substrates.^19–22 Akt activated in this manner is potentially limited by intrinsic feedback circuitry built into this important pathway, prohibiting sustained Akt activation (Fig. 2).²³ Indeed, transient Akt activation early in the replication cycle is observed in primary human fibroblasts infected with wild-type HSV-1 or HCMV (C. McKinney, IM, in preparation).²⁴ Failure to continuously stimulate Akt could limit mTORC1 activation, imposing substantial constraints on viruses that require the host cap-dependent translation machinery and seek to inactivate the 4E-BP1 translational repressor. Some viruses encode multiple functions capable of activating Akt or downstream targets, further illustrating the importance of this task in the virus lifecycle.²² Significantly, TSC-inactivation by Us3 allows HSV1 to activate mTORC1 even when Akt activity is low or undetectable, as may be the case in non-proliferating cells. Furthermore, by acting at the level of TSC, mTORC1 activation by Us3 is not responsive to p70 S6K-mediated cellular feedback controls in place to limit both receptor-mediated activation of the PI3K/Akt/mTORC1 signaling axis and mTORC2-mediated Akt activation (Fig. 2).^23,25 Other viruses likewise activate mTORC1 via TSC, albeit via different mechanism, illustrating the potential advantages of targeting TSC2 to stimulate mTORC1 in infected cells. The related herpesvirus HCMV encodes a TSC2-binding protein (UL38) that inhibits TSC activity, while HPV E6 binds TSC2 and targets it for proteasome degradation.^26,27 None of these strategies, however, involve direct phosphorylation of TSC2 by viral enzymes.Disabling TSC allows viruses to overcome a natural antiviral checkpoint. Interestingly, Us3-deficient virus replication is impaired relative to wild-type in normal primary human fibroblasts and replication is significantly restored upon siRNA-mediated TSC2-depletion. WT virus replication, however, is not impacted by TSC2-depletion, consistent with the observation that TSC is already inactivated in cells infected with WT HSV-1.¹⁷ Thus, TSC comprises an antiviral checkpoint for viruses that require the host cap-dependent translation machinery to produce virus-encoded polypeptides. Replication of viruses that are not equipped to counteract TSC-mediated mTORC1 repression will be limited, unless they bypass this requirement by using an alternative, cap-independent mode of translation initiation that does not require eIF4E.Not only is TSC regulated by Akt, it is also a critical juncture integrating signaling inputs from other pathways. In contrast to Akt, differential TSC2 phosphorylation by AMP-activated protein kinase (AMPK), which is responsive to elevated AMP levels resulting from energy deprivation, stimulates TSC Rheb-GAP activity and prevents mTORC1 activation.²⁸ Induction of the p53-responsive sestrins 1 and 2 in response to genotoxic stress likewise activate AMPK and stimulate TSC Rheb-GAP.²⁹ TSC2 phosphorylation by glycogen synthase kinase 3 cooperates with AMPK-mediated phosphorylation to inhibit mTORC1, linking bioenergetic state with Wnt-signaling responsive GSK3.³⁰ Finally, hypoxia-induced REDD1 activates TSC by interfering with phosphorylation-dependent association of TSC with 14-3-3 proteins.³¹ Binding of phosphorylated TSC2 to 14-3-3 has been proposed to account for Akt-mediated inhibition of TSC. Thus, REDD1-mediated displacement of 14-3-3 from phospho-TSC2 prevents mTORC1 activation even though Akt is constitutively active. In HSV1 infected primary human fibroblasts, Erk activation, which can also phosphorylate TSC2 and inhibit TSC rheb-GAP, is suppressed, making it unlikely that Erk/Rsk signaling plays a role in mTORC1 activation.³ However, how other TSC regulators respond to HSV-1 infection and the potential for Us3 to repress TSC Rheb-GAP activity in response to different stress inputs remains largely unexplored.Given the lack of primary sequence homology between Akt and Us3, it is amazing that Us3 stimulates phosphorylation of Akt substrates other than TSC2 in an Akt-independent manner. Both FOXO1 and GSK3 were among the Akt substrates phosphorylated by Us3 on the same residues targeted by Akt.¹⁷ Thus, Us3 appears to be an Akt surrogate with overlapping substrate specificity that activates mTORC1, stimulating translation and virus replication. As a unique viral kinase unrelated to any single cellular kinase, Us3 is a potential drug development target. Small molecule Us3 inhibitors could prevent HSV1-induced mTORC1 activation and effectively suppress replication without the immune suppressive side-effects associated with targeting mTOR itself.³² The benefits of such a strategy may not be confined to HSV1, given the prevalence of different virus-encoded TSC-inhibitory functions all focused on activating mTORC1 in virus-infected cells.Viruses are masters at encoding multifunctional proteins like Us3, extracting maximum functionality from limited coding regions. Nevertheless, all known Us3 functions are impaired by mutations that eliminate its kinase activity. In addition to the cellular targets TSC2, GSK3 and FOXO1, Us3 has anti-apoptotic activity that likely involves phosphorylation of yet another Akt substrate BAD.^33,34 Us3 also phosphorylates viral proteins some of which stimulate nuclear lamina disassembly and egress of newly assembled progeny virions.^35,36 While not essential for replication, Us3-deficient viruses exhibit cell type-specific replication defects in culture and are severely impaired in mouse pathogenesis models.^37–39 A recently developed cultured rat neuron model of HSV latency/reactivation dependent upon PI-3K/Akt signaling provides an exciting opportunity to probe the role of Us3 in reactivation.⁴⁰ Indeed, by allowing Us3 access to a diverse palate of substrates, a multitude of host and virus-specific tasks can be subverted through the catalytic actions of a single virus enzyme. Given the sheer breadth of these diverse processes involving cellular and viral substrates, Us3 substrate specificity may not be restricted to those of a single cellular kinase like Akt. This raises some important questions regarding the limits of Us3 substrate targeting. For example, are all Akt substrates targeted by Us3, or are some Akt substrates effectively excluded, as their phosphorylation by Us3 may interfere somehow with viral replication? By mimicking Akt activity downstream of the cellular kinase, Us3 could effectively cherry pick which Akt substrates will be phosphorylated and which will be left untouched. Alternatively, the substrate specificity of Us3 could conceivably be so broad that some substrates make little or no detectable contribution to infected cell physiology, representing biological “noise” tolerated but neither advantageous nor detrimental to virus biology. Ultimately, understanding the role of each Us3 substrate in the virus lifecycle will require detailed genetic and biochemical analysis.How Us3 recognizes its full spectrum of substrates, including those that overlap with Akt, remains unknown. Since Us3 bears little resemblance to any specific Ser/Thr kinase, target site prediction is problematic.¹⁷ Studies with synthetic peptide substrates in vitro have not yielded clear-cut results. Although capable of phosphorylating PKA and PKC peptide substrates, Us3 appeared to have a distinct specificity.⁴¹ Sequence recognition motifs, however, are not likely to provide the required in vivo specificity, since (1) many kinases share in vitro recognition motifs (i.e., Arg residues N-terminal to phospho-acceptor site for p90 RSK, PKA and Akt), (2) recognition motifs may not be physiologically phosphorylated, (3) peptides may not mimic intact protein phosphorylation kinetics and (4) not all kinases have a clear consensus motif in peptide substrates.⁴² Instead, interactions beyond the active site that tether the kinase to physiological substrates are often responsible for biological specificity.^43,44 Despite the lack of significant primary sequence homology between Akt and Us3, their functional motifs governing substrate recognition may in fact be related structurally. Such structural similarity in the presence of only limited primary sequence homology has been observed previously among other proteins including globin, lysozyme and thioredoxin family members.^45–47 Finally, the existence of an Akt-like kinase such as Us3 that shares Akt substrates, but not primary sequence homology has potential consequences for the biology of its human host. Is encoding an Akt mimic unrelated at the primary sequence level confined to virus biology, defining an effective strategy to escape from normal constraints that limit Akt activation? Or are there other ways to make kinases with Akt-like substrate specificity lurking in our own genomes? Their unrelatedness at the primary sequence level would render them invisible to present day functional genomic-based identification methods other than pinning them as kinases. Should their identity ever be unmasked, they are likely to play important roles given the critical contributions of Akt signaling to human health and disease.     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

R type anion channel: A multifunctional channel seeking its molecular identity

Plant genomes code for channels involved in the transport of cations, anions and uncharged molecules through membranes. Although the molecular identity of channels for cations and uncharged molecules has progressed rapidly in the recent years, the molecular identity of anion channels has lagged behind. Electrophysiological studies have identified S-type (slow) and R-type (rapid) anion channels. In this brief review, we summarize the proposed functions of the R-type anion channels which, like the S-type, were first characterized by electrophysiology over 20 years ago, but unlike the S-type, have still yet to be cloned. We show that the R-type channel can play multiple roles.Key words: R-type anion channel, nitrate, sulphate, guard cell, action potentialAnion channels play a central role in signal transduction, nutrient transport and cell turgor regulation.¹ By far, their function was particularly well investigated in the guard cells of stomata using a combination of electrophysiological, pharmacological and genetic tools. In this system, anion channel activation was shown to be one of the limiting steps in the loss of cell turgor leading to stomatal closure.² In algal cells, anion channels were shown to contribute to membrane excitability through the generation of action potential.^1,3With the burst of molecular biology in the nineties, the genes coding for plant ion channels started to be unveiled. The first channel gene to be cloned in plant was the shaker-like potassium channel identified in a yeast functional expression screen.^4,5 More than ten years later, TaALMT1 and AtCLCa were characterized as the first members of two important anion channel families.^6,7 This growing group of newly identified channels, accounting for electrophysiological activity described long ago, includes the MSLs anion selective mechanosensitive channels.⁸ Recently, the well known S-type channel has been finally recognized to be encoded by members of the SLAC1 (and other SLAH) family (Slow Anion Channel-Associated 1).⁹ In agreement with electrophysiological data,^10–13 it requires phosphorylation by a Protein Kinase in order to be functional.^14,15 In contrast, the molecular identity of the R-type anion channel remains unknown. Therefore, this candidate, which has been functionally known since twenty years, remains the next challenge for plant channel physiologists.     

Tuba and N-WASP function cooperatively to position the central lumen during epithelial cyst morphogenesis

The process of epithelial lumenogenesis requires coordination of a network of signaling machinery communicated to each cell through subsequent cell divisions. Formation of a single hollow lumen has previously been shown to require Tuba, a Cdc42 GEF, for Cdc42 activation and correct spindle orientation. Using a Caco-2 model of lumenogenesis, we show that knockdown (KD) of the actin regulator N-WASP, causes a multilumen phenotype similar to Tuba KD. Defects in lumenogenesis in Tuba KD and N-WASP KD cells are observed at the two-cell stage with inappropriate marking of the pre-apical patch (PAP )—the precursor to lumen formation. Strikingly, both Tuba and N-WASP depend on each other for localization to the PAP. We conclude that N-WASP functions cooperatively with Tuba to facilitate lumenogenesis and this requires the polyproline region of N-WASP.Key words: lumen, N-WASP, tuba, E-cadherin, pre-apical patchMany epithelial tissues are organized as hollow tubes whose open lumina connect the body with its external environment.^1,2 These tubes consist of a monolayer of polarized cells that envelope the central lumen. Lumen formation is thus a key process in epithelial morphogenesis that depends upon cell polarity to establish three cell surface domains: a basal surface adherent to the extracellular matrix, a lateral surface between cells, and an apical surface that is exposed to the luminal fluids. Of note, the apical membrane is biochemically and morphologically distinct from the baso-lateral surfaces and effectively defines the luminal surface.^3,4For a lumen to form, cells must first mark the site at which apical membrane is to be inserted, something that is achieved at the first cell division.⁵ Targeted trafficking of apical membrane constituents defines a pre-apical patch (PAP), the precursor to the definitive lumen.⁵ Such insertion of apical membrane must presumably be coordinated with the assembly of apical junctions to segregate nascent apical from lateral membrane domains.² Subsequent cell divisions direct apical membrane and protein constituents to this point of initial apical membrane placement.⁶ Coordinated luminal positioning enables the initial formation of a single hollow lumen that subsequently expands through polarized fluid secretion to separate apical membranes, such as occurs in the embryonic gastrointestinal tract,⁷ or by apoptosis or autophagy of the central cells as is observed in mammary gland development.^8,9 Failure to establish initial luminal positioning causes defective lumenogenesis, often resulting in multiple, morphologically abnormal lumina.^5,6Crucial to lumenal morphogenesis is then the mechanism(s) that mark the site where the PAP will form. Cdc42 signaling is increasingly implicated in this process,^2,10 with downstream consequences that include control of mitotic spindle orientation,⁵ which itself influences PAP placement⁵ and potentially regulation of cell-cell junctions. Like other Rho family GTPases, the subcellular location of Cdc42 signaling is determined by the action of upstream proteins, notably guanine nucleotide exchange factors (GEFs).^11,12 Of these, Tuba, a Cdc42-specific GEF,¹³ has emerged as a regulator of lumenal morphogenesis that controls PAP placement through mitotic spindle orientation.¹⁰Tuba is also a scaffolding protein¹³ capable of linking the actin assembly machinery with trafficking pathways. Not only is Tuba required for Cdc42 activation to direct spindle orientation,⁵ it also has the potential to interact with phosphoinositides that define the PAP.¹⁴ Additionally, Tuba binds directly to the actin regulator N-WASP, a key molecule in the organization of actin and itself a Cdc42 effector.¹⁵ Further, Tuba and N-WASP cooperate in various forms of actin-driven cellular motility, such as vesicle propulsion and cell invasive behavior.¹⁶ Interestingly, in epithelial cells N-WASP is also found at cadherin-based cell-cell junctions.¹⁷ In fact it has been proposed that N-WASP functions downstream of Tuba in the maintenance of epithelial junctional homeostasis as N-WASP overexpression was capable of rescuing a Tuba KD phenotype.¹⁸ Therefore, Tuba has the potential to play a central role in coordinating the molecular complexes required for productive polarization of epithelial cells and placement of the PAP during lumenogenesis. However, whether other protein interactions contribute to the morphogenetic impact of Tuba remain to be assessed.Three-dimensional cell culture systems are being utilized to identify critical components in lumen formation. In particular, Madin-Darby canine kidney cells (MDCK) and Caco-2 gastrointestinal cells are commonly used to study cyst and/or tubule formation. MDCK cells undergo both cyst and tubule growth, apoptosis being primarily responsible for the final step in lumen formation,¹⁹ while Caco-2 cells primarily utilize fluid influx to expand cysts.⁵ Cyst culture systems replicate aspects of in vivo organogenesis²⁰ providing tangible, powerful models to analyze and dissect the coordinated cellular mechanisms and processes that occur during epithelial morphogenesis.In this study we examined the relationship between Tuba and N-WASP in early epithelial lumenogenesis using Caco-2 three dimensional cyst cultures. Both Tuba and N-WASP RNAi cell lines result in mature cysts with multiple lumina, and at the two-cell stage, formed multiple PAPs. Interestingly, N-WASP KD perturbed Tuba localization at the PAP, however, N-WASP localization to the PAP was not affected to the same extent by Tuba KD. Taken together, these results suggest a complex interrelationship between Tuba and N-WASP for the coordinated formation of a single hollow lumen.